捕食线虫真菌对松材线虫捕食率测定的新方法

本文依据捕食线虫真菌的捕食特性以及松材线虫的生长习性,对松材线虫进行了定时,定量的测定试验,试图通过培养后线虫的种群数量变化,来摸索定量测定捕食率的方法.在漏斗法基础上进行了相应的改进,成功地摸索出3种方法捕食率测定的新方法.各方法各具优缺点,滤膜镜检法和双层镜检法的数据更精确,条切镜检法和漏斗法所测数值较粗略.因此,可依据实验的侧重点以及实验所需的精密程度,而采用最适宜的方法进行捕食率测定实验.     

Comparative Bioactivity of Emamectin Benzoate Formulations against the Pine Wood Nematode,Bursaphelenchus xylophilus

The pine wood nematode (PWN), Bursaphelenchus xylophilus is a well-known devastating pathogen of economic importance in the Republic of Korea and other countries. In the Republic of Korea, trunk injection of nematicides is the preferred method of control. In this study, the efficacy of 16 locally produced formulations of emamectin benzoate against the PWN are compared through determining their sublethal toxicities and reproduction inhibition potentials. Nematodes were treated with varying concentrations of the tested chemicals in multi-well culture plates, and rates of paralysis and mortality were determined after 24 h. Reproduction inhibition potential was tested by inoculating pre-treated nematodes onto Botrytis cinerea, and in pine twig cuttings. Despite the uniformity in the concentration of the active ingredient, efficacy was contrastingly different among formulations. The formulations evidently conformed to three distinct groups based on similarities in sublethal activity (group 1: LC₉₅ of 0.00768–0.01443 mg/ml; group 2: LC₉₅ of 0.03202–0.07236 mg/ml, and group 3: LC₉₅ of as high as 0.30643–0.40811 mg/ml). Nematode paralysis generally occurred at the application dose of 0.0134–0.1075 μg/ml, and there were significant differences in nematode paralysis rates among the products. Nematode reproduction was only evident at lower doses both on B. cinerea and pine twigs, albeit the variations among formulations. Group 1 formulations significantly reduced nematode reproduction even at a lower dose of 0.001075 μg/ml. The variations in efficacy might be attributed to differences in inert ingredients. Therefore, there is need to analyze the potential antagonistic effects of the large number of additives used in formulations.     

Effects of Bacteria Associated with Pine Wood Nematode (Bursaphelenchus xylophilus) on Development and Egg Production of the Nematode

We tested the effects of four bacterial strains carried on the surface of the pine wood nematode (PWN), Bursaphelenchus xylophilus, on egg hatch, development rate and egg production. Strains GcM5‐1A (Pseudomonas fluorescens) and ZpB1‐2A (P. putida), were strong phytotoxin producers, while strains JnB1B (Pantoea sp.) and AcB1C (Peptostreptococcus asaccharalyticus) did not produce phytotoxins. None of the strains had any effect on egg hatch. GcM5‐1A and ZpB1‐2A promoted egg production, developmental rate, body length and diameter growth in both male and female PWN, whereas JnB1B and AcB1C had no such effects on the nematode. Indeed, the latter two strains completely inhibited egg production of the nematode. The results suggest that GcM5‐1A and ZpB1‐2A may provide PWN with food and/or essential nutrients for development and egg production. These results provide further evidence for our previous finding of a mutualistic symbiosis between the PWN and certain strains of bacteria carried by this nematode ( Zhao et al., 2003, 2005 ).     

Observations on the Morphology of the Pine Wood Nematode, Bursaphelenchus xylophilus

Scans of slash pine wood chips infested with pine wood nematodes showed coiled aggregates of 5-10 nematodes in the axial resin canals, and 1-2 nematodes folded in the radial resin canals. Observations of the pine wood nematode by SEM showed the head to be offset with six labial lobes. A smaller circle of six inner lobes surrounded the stoma. A single labial sensillum was observed on each of the two subventral and two subdorsal lobes. The two opposite lobes, without sensilla, bore a pore-like amphid. The female vulva flap was observed to be a pouch. In addition to two pairs of copulatory papillae previously described for the male, three additional papillae were observed: an additional post-anal pair, and a single papilla anterior to the cloaca. Spicules were fused ventrally. Distal spicule ends terminated in two fused half discs. Caudal alae were angular at the tail terminus, but culed inward at the corners to appear oval. Lateral fields had four lateral striations.     

Scanning Electron Microscopy of Pine Seedling Wood Tissue Sections Inoculated with the Pinewood Nematode Bursaphelenchus xylophilus Previously Prepared for Light Microscopy

Scanning electron microscopy (SEM) was applied to paraffin-embedded wood sections to study the histopathology of pine seedlings inoculated with the pinewood nematode (PWN), Bursaphelenchus xylophilus. The sections, which had been previously prepared and observed by light microscopy (LM) on glass slides, were originally obtained from experiments in which pine seedlings had been inoculated with PWN. The cover glass was removed by soaking the glass slide in xylene for 3 to 5 days. The glass slides were cut into small pieces so that each piece contained one wood section. Each piece of the glass slide was attached with double adhesive tape to an aluminum stub. The specimens were sputter-coated with gold and examined with a scanning electron microscope (JEOL-JSM 5200). Compared to LM (as documented in previous reports) SEM provided greater depth of focus and resolution of the damaged wood tissues, nematodes and associated bacteria. SEM made it possible to observe the relationship between bacterial distribution and nematode distribution in wood tissues. SEM observations also suggested the possibility of documenting the death of ray cells and other parenchyma cells in relation to disease development. Finally, the current study of PWN in pine seedlings demonstrated that glass slides prepared for LM observations more than 25 years earlier could be successfully processed for examination by SEM.     

Pathotypes of the Pinewood Nematode Bursaphelenchus xylophilus

An isolate of Bursaphelenchus xylophilus from Pinus sylvestris in Missouri infected and reproduced in 2-3-year-old seedlings of P. sylvestris and to some extent in seedlings of P. nigra. Wilting, however, occurred only in P. sylvestris. B. xylophilus isolated from P. strobus in Vermont infected and reproduced only in P. strobus seedlings. P. taeda seedlings were resistant to both of these isolates. Phytotoxin production was seen only in susceptible seedling species-nematode combinations. Significant water loss occurred only in those seedlings that were wilted because of infection by a compatible nematode isolate. Our results suggest that these isolates are pathotypes of B. xylophilus.     

Lethal Temperature for Pinewood Nematode, Bursaphelenchus xylophilus, in Infested Wood Using Microwave Energy

To reduce the risks associated with global transport of wood infested with pinewood nematode Bursaphelenchus xylophilus, microwave irradiation was tested at 14 temperatures in replicated wood samples to determine the temperature that would kill 99.9968% of nematodes in a sample of ≥ 100,000 organisms, meeting a level of efficacy of Probit 9. Treatment of these heavily infested wood samples (mean of > 1,000 nematodes/g of sapwood) produced 100% mortality at 56 °C and above, held for 1 min. Because this "brute force" approach to Probit 9 treats individual nematodes as the observational unit regardless of the number of wood samples it takes to treat this number of organisms, we also used a modeling approach. The best fit was to a Probit function, which estimated lethal temperature at 62.2 (95% confidence interval 59.0-70.0) °C. This discrepancy between the observed and predicted temperature to achieve Probit 9 efficacy may have been the result of an inherently limited sample size when predicting the true mean from the total population. The rate of temperature increase in the small wood samples (rise time) did not affect final nematode mortality at 56 °C. In addition, microwave treatment of industrial size, infested wood blocks killed 100% of > 200,000 nematodes at ≥ 56 °C held for 1 min in replicated wood samples. The 3(rd)-stage juvenile (J3) of the nematode, that is resistant to cold temperatures and desiccation, was abundant in our wood samples and did not show any resistance to microwave treatment. Regression analysis of internal wood temperatures as a function of surface temperature produced a regression equation that could be used with a relatively high degree of accuracy to predict internal wood temperatures, under the conditions of this study. These results provide strong evidence of the ability of microwave treatment to successfully eradicate B. xylophilus in infested wood at or above 56 °C held for 1 min.     

Within-wood Spatial Dispersion of the Pinewood Nematode,Bursaphelenchus xylophilus

Pinewood nematode, Bursaphelenchus xylophilus (Steiner &Buhrer) Nickle, spatial dispersion was determined in Scots pine, Pinus sylvestris L., bolts infested with the pine sawyer beetle, Monochamus carolinensis (Olivier) and in bolts without M. carolinensis. According to Taylor''s power law and Green''s index of dispersion, nematode dispersion was aggregated in both sets of bolts. The degree of aggregation did not differ significantly between beetle-infested and noninfested bolts, suggesting that the presence of M. carolinensis does not affect nematode dispersion within a bolt. Nematode population densities differed radially in bolts not infested with pine sawyers, but in a nonregular pattern. Moisture content of the bolts was correlated with population density of B. xylophilus, suggesting that nematode aggregates occur in areas of high moisture content.     

Bacterial Diversity and Community Structure in the Pine Wood Nematode Bursaphelenchus xylophilus and B. mucronatus with Different Virulence by High-Throughput Sequencing of the 16S rDNA

Bursaphelenchus xylophilus is the pathogen of pine wilt disease. Bursaphelenchus mucronatus is similar to B. xylophilus in morphology. Both species share a common niche, but they are quite different in pathogenicity. Presently, the role of bacteria in pine wilt disease development has been widely speculated. The diversity of bacteria associated with B. xylophilus and B. mucronatus with different virulence remains unclear. In this study, virulence of four B. xylophilus and four B. mucronatus strains were evaluated by inoculating Pinus thunbergii. High-throughput sequencing targeted 16S rDNA of different virulence nematode strains was carried out. The associated bacterial community structures of the eight strains were analyzed. The results showed that 634,051 high-quality sequences were obtained from the eight nematode strains. The number of OTUs of bacteria associated with B. mucronatus was generally greater than those of B. xylophilus. The richness of the community of bacteria associated with high virulent B. xylophilus ZL1 and AmA3 was higher than moderately virulent B. xylophilus AA3, HE2, and all B. mucronatus strains. While the diversity of bacteria associated with B. mucronatus was higher than B. xylophilus. Stenotrophomonas, Pseudomonadaceae_Unclassified or Rhizobiaceae_Unclassified were predominant in the nematode strains with different virulence. Oxalobacteraceae and Achromobacter were found more abundant in the low virulent B. xylophilus and non-virulent B. mucronatus strains.     

Specific and Functional Diversity of Endophytic Bacteria from Pine Wood Nematode Bursaphelenchus Xylophilus with Different Virulence

Pine wilt disease (PWD) caused by the pine wood nematode (PWN), Bursaphelenchus xylophilus, is one of the most devastating diseases of Pinus spp. The PWN was therefore listed as one of the most dangerous forest pests in China meriting quarantine. Virulence of the PWN is closely linked with the spread of PWD. However, main factors responsible for the virulence of PWNs are still unclear. Recently epiphytic bacteria carried by PWNs have drawn much attention. But little is known about the relationship between endophytic bacteria and virulence of B. xylophilus. In this research, virulence of ten strains of B. xylophilus from different geographical areas in six provinces of China and four pine species were tested with 2-year-old seedlings of Pinus thunbergii. Endophytic bacteria were isolated from PWNs with different virulence to investigate the relationship between the bacteria and PWN virulence. Meanwhile, the carbon metabolism of endophytic bacteria from highly and low virulent B. xylophilus was analyzed using Biolog plates (ECO). The results indicated that ten strains of PWNs showed a wide range of virulence. Simultaneously, endophytic bacteria were isolated from 90% of the B. xylophilus strains. The dominant endophytic bacteria in the nematodes were identified as species of Stenotrophomonas, Achromobacter, Ewingella, Leifsonia, Rhizobium, and Pseudomonas using molecular and biochemical methods. Moreover, S. maltophilia, and A. xylosoxidans subsp. xylosoxidans were the predominant strains. Most of the strains (80%) from P. massoniana contained either S. maltophilia, A. xylosoxidans, or both species. There was a difference between the abilities of the endophytic bacteria to utilize carbon sources. Endophytic bacteria from highly virulent B. xylophilus had a relatively high utilization rate of carbohydrate and carboxylic acids, while bacteria from low virulent B. xylophilus made better use of amino acids. In conclusion, endophytic bacteria widely exist in B. xylophilus from different pines and areas; and B. xylophilus strains with different virulence possessed various endophytic bacteria and diverse carbon metabolism which suggested that the endophytic bacteria species and carbon metabolism might be related with the B. xylophilus virulence.     

On the Taxonomy and Morphology of the Pine Wood Nematode, Bursaphelenchus xylophilus (Steiner &Buhrer 1934) Nickle 1970

During the past 3 yr, nematologists in the United States have found specimens of Bursaphelenchus sp. in the wood of dead and dying pine trees. This nematode-host association resembles a similar interaction reported from Japan where pine trees are being killed by the pine wood nematode. This taxonomic research was conducted to determine if the Japanese pine wood nematode and similar populations in the United States are of the same species. Based upon typical morphological characters of original specimens of Bursaphelenchus xylophilus (Steiner and Buhrer 1934) Nickle 1970 that were rediscovered in the USDA Nematode Collection and genetic crosses among the Japanese and American nematode populations, it was concluded that they are all the same species, B. xylophilus.     

Cultivation of the Pine Wilt Nematode, Bursaphelenchus xylophilus,in Axenic Culture Media

The pine wilt nematode, Bursaphelenchus xylophilus, has been cultured axenically in vitro on soy peptone/yeast extract or modified Caenorhabditis medium supplemented with cholesterol and hemoglobin. Although growth, development and reproduction were best in soy peptone/yeast extract medium, satisfactory population size increases were observed in the chemically defined Caenorhaditis medium.     

Molecular Characterization and Development of Real-Time PCR Assay for Pine-Wood Nematode Bursaphelenchus xylophilus (Nematoda: Parasitaphelenchidae)

Bursaphelenchus xylophilus, the pine-wood nematode (PWN), is the causal agent of pine wilt disease, one of the most damaging emerging pest problems to forests around the world. It is native to North America where it causes relatively minor damage to native conifers but is labeled an EPPO-A-2 pest and a quarantine nematode for many countries outside of the United States because of its potential for destruction to their native conifers. Exports of wood logs and commodities involving softwood packaging materials now require a lab test for the presence/absence of this regulated nematode species. We characterized the DNA sequences on the ribosomal DNA small subunit, large subunit D2/D3, internal transcribed spacer (ITS) and mitochondrial DNA cytochrome oxidase subunit one on the aphelenchid species and described the development of a real-time-PCR method for rapid and accurate identification of PWN targeting the ITS-1. A total of 97 nematode populations were used to evaluate the specificity and sensitivity of this assay, including 45 populations of B. xylophilus and 36 populations of 21 other species of Bursaphelenchus which belong to the abietinus, cocophilus, eggersi, fungivorus, hofmanni, kevini, leoni, sexdentati, and xylophilus groups and one unassigned group from a total of 13 groups in the genus Bursaphelenchus; 15 populations of Aphelenchoides besseyi, A. fragariae, Aphelenchoides species and Aphelenchus avenae; and one population of mixed nematode species from a soil sample. This assay proved to be specific to B. xylophilus only and was sensitive to a single nematode specimen regardless of the life stages present. This approach provides rapid species identification necessary to comply with the zero-tolerance export regulations.     

Efficacy of Four Nematicides Against the Reproduction and Development of Pinewood Nematode,Bursaphelenchus xylophilus

To understand the efficacy of emamectin benzoate, avermectin, milbemectin, and thiacloprid on the reproduction and development of Bursaphelenchus xylophilus, seven parameters, namely population growth, fecundity, egg hatchability, larval lethality, percent larval development, body size, and sexual ratio, were investigated using sublethal (LC₂₀) doses of these compounds in the laboratory. Emamectin benzoate treatment led to a significant suppression in population size, brood size, and percent larval development with 411, 3.50, and 49.63%, respectively, compared to 20850, 24.33, and 61.43% for the negative control. The embryonic and larval lethality increased obviously from 12.47% and 13.70% to 51.37% and 75.30%, respectively. In addition, the body length was also significantly reduced for both males and females in the emamectin benzoate treatment. Avermectin and milbemectin were also effective in suppressing population growth by increasing larval lethality and reducing larval development, although they did not affect either brood size or embryonic lethality. Body length for both male and female worms was increased by avermectin. Thiacloprid caused no adverse reproductive effects, although it suppressed larval development. Sexual ratio was not affected by any of these four nematicides. Our results indicate that emamectin benzoate, milbemectin, and avermectin are effective against the reproduction of B. xylophilus. We think these three nematicides can be useful for the control of pine wilt disease.     

New Insights into the Phylogeny and Worldwide Dispersion of Two Closely Related Nematode Species,Bursaphelenchus xylophilus and Bursaphelenchus mucronatus

The pinewood nematode, Bursaphelenchus xylophilus, is one of the greatest threats to coniferous forests worldwide, causing severe ecological damage and economic loss. The biology of B. xylophilus is similar to that of its closest relative, B. mucronatus, as both species share food resources and insect vectors, and have very similar morphological characteristics, although little pathogenicity to conifers has been associated with B. mucronatus. Using both nuclear and mitochondrial DNA markers, we show that B. xylophilus and B. mucronatus form distinct phylogenetic groups with contrasting phylogeographic patterns. B. xylophilus presents lower levels of intraspecific diversity than B. mucronatus, as expected for a species that evolved relatively recently through geographical or reproductive isolation. Genetic diversity was particularly low in recently colonised areas, such as in southwestern Europe. By contrast, B. mucronatus displays high levels of genetic diversity and two well-differentiated clades in both mitochondrial and nuclear DNA phylogenies. The lack of correlation between genetic and geographic distances in B. mucronatus suggests intense gene flow among distant regions, a phenomenon that may have remained unnoticed due to the reduced pathogenicity of the species. Overall, our findings suggest that B. xylophilus and B. mucronatus have different demographic histories despite their morphological resemblance and ecological overlap. These results suggest that Bursaphelenchus species are a valuable model for understanding the dispersion of invasive species and the risks posed to native biodiversity and ecosystems.     

Manipulation of Auxin Transport in Plant Roots during Rhizobium Symbiosis and Nematode Parasitism

The plant rhizosphere harbors many different microorganisms, ranging from plant growth–promoting bacteria to devastating plant parasites. Some of these microbes are able to induce de novo organ formation in infected roots. Certain soil bacteria, collectively called rhizobia, form a symbiotic interaction with legumes, leading to the formation of nitrogen-fixing root nodules. Sedentary endoparasitic nematodes, on the other hand, induce highly specialized feeding sites in infected plant roots from which they withdraw nutrients. In order to establish these new root structures, it is thought that these organisms use and manipulate the endogenous molecular and physiological pathways of their hosts. Over the years, evidence has accumulated reliably demonstrating the involvement of the plant hormone auxin. Moreover, the auxin responses during microbe-induced de novo organ formation seem to be dynamic, suggesting that plant-associated microbes can actively modify their host''s auxin transport. In this review, we focus on recent findings in auxin transport mechanisms during plant development and on how plant symbionts and parasites have evolved to manipulate these mechanisms for their own purposes.     

Incubation Temperature and Time Effects on Life Stages of Bursaphelenchus xylophilus in Wood Chips

Wood chips of Pinus strobus inoculated with Bursaphelenchus xylophilus were incubated at 3, 12, 30, or 40 C during intervals of 47, 82, and 130 days to determine the effects of incubation temperature and time on total number of nematodes and occurrence of each life stage. Nematodes did not survive at 40 C; the greatest number of nematodes was maintained at 3 C. The number and percentage of juveniles in the propagative cycle were greatest at 3 C after 47 days, but the percentage was greatest at 30 C after 130 days. More third-stage dispersal larvae, with percentages as high as 85%, were extracted at 3 and 12 C than at 30 C by the end of the study. Dauer larvae were extracted from the chips, but percentages never exceeded 5%. The percentage of adults was greater at 30 C than at 3 and 12 C after 82 and 130 days. When a 1-week heat treatment at 30 C was applied to samples at 3 and 12 C,. numbers and percentages of adults increased. Percentages of dauer larvae increased very slightly when the heat treatment was applied after 47 days, but numbers and percentages of juveniles and dispersals were affected erratically.     

Effect of Monochamus carolinensis on the Life History of the Pinewood Nematode,Bursaphelenchus xylophilus

The development of Bursaphelenchus xylophilus in pine wood infested with and free of Monochamus carolinensis was investigated. Formation of third-stage dispersal juveniles occurred in the presence and absence of pine sawyer beetles. The proportion of third-stage dispersal juveniles in the total nematode population was negatively correlated with moisture content of the wood. Formation of nematode dauer juveniles was dependent on the presence of the pine sawyer beetle. Dauer juveniles were present in 3 of 315 wood samples taken from non-beetle-infested Scots pine bolts and 81 of 311 samples taken from beetle-infested bolts. Nematode densities were greater in wood samples taken adjacent to insect larvae, pupae, and teneral adults compared with samples taken from areas void of insect activity. Nematodes recovered from beetle larvae, pupae, and teneral adults were mostly fourth-stage dauer juveniles, although some third-stage dispersal juveniles were also recovered. Dauer juvenile density was highest on teneral adult beetles.